Survival outcomes remain very poor for lung cancer patients, in part due to treatment resistance. K-Ras mutations are among the common genetic alterations in human lung cancer. These genetic aberrations may negatively affect treatment response to chemoradiotherapy. Bcl2 is the major anti-apoptotic member of the Bcl2 family that may act as downstream survival substrates of these genetic mutations-activated signaling pathway(s). Our preliminary data indicate that genetic alterations in K-Ras, ionizing radiation (IR) and mTOR inhibition by rapalog positively regulate expression and/or phosphorylation of Bcl2 in lung cancer cells or NSCLC patients, which could contribute to radioresistance. Development of novel small molecule compounds to counteract activation of Bcl-2 induced by K-Ras mutations, IR or rapalog should reverse radio- or rapalog resistance leading to improvement of lung cancer outcome. Since the BH4 survival domain is required for Bcl2's antiapoptotic function, small molecules that interfere with the BH4 domain represent a novel strategy to disrupt the antiapoptotic function of Bcl2. We chose the BH4 domain of Bcl2 as docking site to screen small molecules using the UCSF DOCK 6.1 program suite and the NCI chemical library database. We identified four compounds with activity against lung cancer and named them small molecule Bcl2 BH4 domain antagonists (i.e. BDAs). According to the potency and drug-likeness, we chose BDA-366 as the lead for further investigation in this proposal. BDA-366 potently represses lung cancer without significant normal tissue toxicity in vivo. Since increased levels of Bcl2 and mTOR were observed in radio- or rapalog resistant lung cancer cells, we hypothesize that BDA-366 or in combination with mTOR inhibitor may represent an optimal strategy for overcoming radio- or rapalog resistance. K-Ras mutations-activated MEK/ERK1/2 can induce phosphorylation of Bcl2. We found that increased levels of phospho-Bcl2 (pBcl2) in tumor tissues are associated with poor prognosis of NSCLC patients. We hypothesize that pBcl2 may provide new predictive and prognostic biomarker in NSCLC. In this proposal, three specific aims have been identified: (1) To determine whether and how K-Ras mutations, radiation or mTOR inhibition positively regulate Bcl2 expression and phosphorylation leading to radio- or rapalog resistance in human lung cancer cells; (2) To determine the association between K-Ras mutation(s) and Bcl2 phosphorylation and whether this association defines clinical subsets of NSCLC; (3) To determine mechanism by which novel small molecule Bcl2 BH4 antagonist (BDA-366) inactivates Bcl2 and induces apoptosis. Studies will test the potency of BDA-366 in the resistant lung cancer and the genetically engineered lung cancer animal models. Proposed studies will uncover novel genetic signaling pathways and identify new biomarker relevant for lung cancer treatment. It is expected that a new class of novel agents will be developed, which can overcome both radio- and rapalog resistances in NSCLC.